One method for cells receiving signals from the external environment is through ligand-receptor interactions. In one scenario, the receptor is integral to the cell and is embedded within the plasma membrane bilayer of the cell. The receptor may traverse the entire bilayer or reside within the layer having a portion of it exposed on the surface of the cell. Typical ligands that interact with these receptors are hydrophilic molecules. Through this interaction, secondary events are triggered leading to changes within the cytosol of the cell such as protein phosphorylation.
In contrast to hydrophilic ligands, hydrophobic molecules, such as steroid and thyroid hormones, pass through the plasma membrane of a cell and interact with specific receptor proteins residing within the cytoplasm or nucleus. Other hydrophobic molecules, such as retinoic acid, are metabolically synthesized in the cell itself. Similarly to externally derived hydrophobic molecules, hydrophobic compounds that are synthesized intracellularly interact with intracellular receptor proteins to exert their biological effects.
For example, steroid hormones (testosterone, estrogen, etc.) dissociate from plasma-binding proteins and cross the plasma membrane and enter target cells. Steroid hormone receptors are tissue-specific binding proteins found in low concentrations in the cytoplasm of the cell. When steroid receptors are occupied by ligand they change conformation and become activated with enhanced affinity for nuclear chromatin. The activated hormone-receptor complex accumulates in the nucleus bound to chromosomal DNA containing acceptor sites for the complex. The high affinity interaction of the steroid-hormone receptor complex with nuclear chromatin results in activation of DNA transcription and in the synthesis of specific mRNAs.
Hydrophobic ligands other than steroids similarly bind to and activate different nuclear receptors. For example, the receptors for thyroid hormones are found in the nucleus even in the absence of their ligand. Thyroid hormones enter cells and travel to the nucleus. Specific genes are under thyroid hormone control, and they are transcribed to particular mRNA in response to this ligand. In turn, translation of the mRNA results in the synthesis of specific cell proteins.
In addition to nuclear receptors, hydrophobic ligands bind in cells to proteins that are members of a family of homologous proteins termed intracellular lipid binding proteins (iLBPs). These proteins reside in the cytosol of cells. Some iLBPs move to the nucleus when they bind their cognate ligands. For example, the iLBP called adipocyte fatty acid binding protein (adipocyte FABP) moves from the cytosol to the nucleus following binding of its cognate ligands. Intracellular lipid binding proteins often share ligands with particular nuclear receptors. For example, the anti-diabetic drug troglitazone binds to the nuclear receptor termed peroxisome proliferator activated receptor γ (PPARγ) and also associates with adipocyte FABP.
Another example of nuclear receptor ligands are the vitamin A metabolites retinoic acids (“RA”) and their synthetic derivatives, collectively known as retinoids, which can be used in the treatment of a variety of pathologies ranging from dermatological disorders to cancer.
The retinoid members of the nuclear hormone receptor superfamily are responsive to compounds referred to as retinoids, which include retinoic acid and a series of natural and synthetic derivatives which have been found to exert profound effects on development and differentiation in a wide variety of systems.
Retinoic acid-dependent transcription factors, referred to as RARs (retinoic acid receptors), have been identified. Currently, three different RAR subtypes (alpha, beta and gamma) and several isoforms of each are known to exist in mammals. RARs share sequence homology with other members of the superfamily of nuclear hormone receptors. This family of proteins encompasses ligand-dependent transcription factors that regulate the expression of particular target genes upon binding of specific ligands. Different RAR subtypes are expressed in distinct patterns throughout development and in the mature organism.
Additional members of the nuclear hormone superfamily of receptors that respond to retinoids have been identified. These are termed retinoid X receptors (RXRs): RXR-α (see Mangelsdorf et al., in Nature 345: 224-229 (1990)), RXR-β (see Hamada et al., Proc. Natl. Acad. Sci. USA 86: 8289-8293 (1989)), and RXR-γ (see Mangelsdorf et al., Genes and Development 6:329-344 (1992)).
Although both RARs and RXRs respond to retinoic acids, these receptors differ in several important aspects. First, RAR and RXR are significantly divergent in primary structure. These sequence differences are reflected in differential responsiveness of RAR and RXR to various vitamin A metabolites and synthetic retinoids. In addition, distinctly different patterns of tissue distribution are seen for RAR and RXR. Furthermore, while RXR can activate transcription as a homodimer, i.e. on its own, the transcriptional activity of RAR is mediated through RAR-RXR heterodimers. Finally, RXR homodimers bind to response elements that are distinct from the DNA sequences that are recognized by RAR-RXR heterodimers, and thus RXR-RXR and RXR-RAR complexes regulate the expression of different genes.
Retinoid therapy is complicated by the toxicity of these compounds at pharmacological doses. Existing methods for retinoid detection and quantification consist of organic solvent extractions and HPLC analyses, procedures that are too time-consuming and expensive to be used in the hospital/clinic setting. Consequently, as currently practiced, retinoid treatment is not individualized for particular patients but is administered by ‘standard’ dosing. This is so despite the high toxicity of these compounds and the large patient-to-patient variability in resulting plasma concentrations of RA.
Certain diseases affect or are affected by processes that alter physiological events that are associated with specific ligand-receptor interactions. Clearly, the detection and quantitation of ligands that bind to nuclear receptors is important diagnostically as well as for monitoring physiological effects during a treatment regime.